An accessible and high-throughput strategy of continuously monitoring apoptosis by fluorescent detection of caspase activation

Abstract

We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time.

Modelling and Prevention of Acute Kidney Injury through Ischemia and Reperfusion in a Combined Human Renal Proximal Tubule/Blood Vessel-on-a-Chip

“…DNA staining (Fig. 3b) and activated caspase 3/7 staining [44] (Fig. 3c) confirmed these observations, showing visible damage for concentration of 27 µM cisplatin or 28.1 mM tobramycin and higher.…”

“…DNA staining (Figure 3B) and activated caspase-3/7 staining (44) (Figure 3C) confirmed these observations, showing visible damage for concentration of 27- µ M cisplatin or 28.1-mM tobramycin and higher. Tubules exposed to the highest concentration of CysA showed a slight increase in the number of positive caspase-3/7 cells, although this effect was less dominant compared with the other two compounds.…”

Ibuprofen and diclofenac differentially affect cell viability, apoptosis and morphology changes of human cholangiocarcinoma cell lines

“…Caspase 3/7 activity and Annexin V-FITC/PI assays were used to confirm cell apoptosis with a microplate reader and fluorescence detection of caspase activity, as previously described. 20 , 21 Annexin V-FITC/PI staining is used to detect apoptosis on the basis of staining of phosphatidylserine molecules that have translocated outside the cell membrane. Whereas viable cells with intact membranes exclude PI, the membranes of dead and damaged cells are permeable to PI.…”

Cholesterol biosynthesis modulates differentiation in murine cranial neural crest cells

“…Real-time, automated Incucyte live-cell Casp3/7 apoptosis assays 62 were performed on cultures that were ~ 30% confluent. The assays were conducted using an Incucyte live-cell analysis system (Sartorius) via direct treatment with Incucyte Caspase-3/7 dyes, monitoring by time-lapse imaging (every 2 h for 3d), and apoptosis quantification using the Incucyte Cell-by-Cell Analysis Software Module 63 (Essen Bioscience).cNCC migration was examined using the Incucyte Scratch Wound Assay 64 .…”